The invention relates to solid forms of antibodies against the EGF receptor (EGFR), in particular precipitates and crystals of monoclonal antibodies against the EGF receptor, particularly preferably of Mab C225 (cetuximab) and Mab h425 (EMD 72000), which result in biologically active antibody protein through dissolution or suspension in aqueous or non-aqueous medium, obtainable by precipitation of the antibody and/or one of its variants and/or fragments dissolved or suspended in aqueous medium by means of a precipitation reagent. The invention furthermore relates to pharmaceutical preparations comprising at least one solid form of the above-mentioned antibodies in precipitated non-crystalline, precipitated crystalline or in dissolved or suspended form, and optionally excipients and/or adjuvants and/or further pharmaceutical active ingredients, and to a process for the preparation of solid forms of anti-EGFR antibodies according to the invention.
Advances in the area of biotechnology have made it possible in the course of the last 10 years to prepare a series of proteins for pharmaceutical application by means of recombinant DNA techniques. Protein medicaments, such as monoclonal antibodies, are used, for example, in tumour therapy, for example for specific immunotherapy or tumour vaccination. Therapeutic proteins are larger and more complex than conventional organic and inorganic active ingredients and they have complex three-dimensional structures and numerous functional groups which effect the biological activity of the protein or alternatively can cause undesired effects. During preparation, storage and transport, protein medicaments are exposed to numerous exogenous influences which can have a stability-reducing action on the protein active ingredient. It is therefore necessary to study accurately the causes and mechanisms of the specific degradation reactions in order to be able to stabilise the protein, for example through addition of certain stabilising adjuvants (see, for example, Manning M. C., Patel K., & Borchardt R. T. (1989) Stability of protein pharmaceuticals. Pharm. Res. 6, 903-918).
The literature discloses numerous formulations of therapeutic proteins. However, the requirements of the composition of a pharmaceutical preparation of protein active ingredients may be very different, and in general it is not possible, owing to specific physico-chemical properties and degradation reactions of the different proteins, to apply already established protein formulations to novel protein active ingredients. Suitable pharmaceutical formulations and stable forms of these novel active ingredients are therefore still a major challenge.
Chemical instabilities are distinguished by covalent modifications of the protein. The primary structure of the protein changes through the breaking, new formation or re-formation of chemical bonds. The newly formed substance is generally completely different in biological activity from the original, native protein. Physical instabilities modify the spatial arrangement of the molecule (the secondary, tertiary and quaternary structure) without destroying covalent bonds. They can be divided into denaturing, association, aggregation, precipitation or adsorption. Physical instabilities are a frequent phenomenon, in particular in the case of relatively large proteins. Precipitates are the macroscopically visible equivalent of aggregates and are formed in mechanistic terms by clusters of aggregates or associates. By exceeding the solubility limit and due to precipitation, the flakes become visible from a diameter of about 10 μm through a light microscope and from about 50 μm with the naked eye. Protein aggregation can be a reversible or irreversible process (see, for example, Cleland J. L., Powell M. F. & Shire S. J. (1993) The development of stable protein formulations: A close look at protein aggregation, deamidation, and oxidation. Crit. Rev. Ther. Drug Carrier Syst. 10, 307-377).
Although the previous literature describes the precipitation of proteins with salts, polymers and organic solvents as standard method for the purification of proteins (Scopes R. K. (1997) Separation by Precipitation. In: Protein Purification: Principles and Practice (ed Scopes R. K.), 2 edn, pp. 41-71. Springer Verlag, N.Y.), the use of this method usually results, however, particularly in the case of immunoglobulins, in denaturing, an associated reduction in activity and in poor quantitative yields, in particular on use of salts and organic solvents (Phillips A. P., Martin K. L., & Horton W. H. (1984) The choice of methods for immunoglobulin IgG purification: Yield and purity of antibody activity. Journal of Immunological Methods 74, 385-393). On use of polyethylene glycol (PEG), by contrast, better results are achieved (A. Poison, G. M. Potgieter, J. F. Largier, and G. E. F. Joubert, F. J. Mears. The Fractionation of Protein Mixtures by linear Polymers of High Molecular Weight. Biochim. Biophys. Acta 82:463-475, 1964).
Protein crystals are known from purification processes (downstream processing), preferably of enzymes, and for the elucidation of the tertiary structure of proteins by means of X-ray structural analysis (R. K. Scopes. Analysis for purity: Crystallization. In: Protein Purification: Principles and Practice, edited by R. K. Scopes, New York:Springer Verlag, 1997, p. 284-301). The formation of new ordered intermolecular contacts between proteins occurs here. This is a slow process, with reduced mobility. The concentration of the protein in solution is reduced in the process.
Although the literature describes the crystallisation of proteins with salts, polymers and organic solvents as standard method for elucidation of the structure of immunoglobulins (Harris L. J., Skaletsky E., & McPherson A. (1995) Crystallization of Intact Monoclonal Antibodies. Proteins: Structure, Function, and Genetics 23, 285-289; Harris L. J., Skaletsky E., & McPherson A. (1998) Crystallographic Structure of an Intact Ig1 Monoclonal Antibody. Journal of Molecular Biology 275, 861-872; Edmundson A. B., Guddat L. W., & Andersen K. N. (1993) Crystal Structures of intact IgG antibodies. ImmunoMethods 3, 197-210), the crystallisation of intact, for example glycosylated antibodies is, however, extremely difficult since the size of the protein, the different glycosylation pattern of the individual anti-body molecules and the associated microheterogeneities as well as the structural flexibility of the immunoglobulin make ordered incorporation into a crystal lattice more difficult or even prevent it (McPherson A. (1999) Crystallization of Biological Macromolecules, 1 edn. Cold Spring Harbor Laboratory Press, New York). In addition, antibody molecules exhibit a tendency towards aggregation, which likewise causes great difficulties in crystallisation (McPherson A. (1999) Crystallization of Biological Macro-molecules, 1 edn. Cold Spring Harbor Laboratory Press, New York). In addition, the risk of denaturing of the antibodies during the crystallisation process makes the crystallisation of therapeutic antibodies unattractive to the person skilled in the art. Thus, only a few intact antibodies have hitherto been crystallised for structural elucidation and only three antibodies have hitherto been crystallised on a preparative scale. Thus, the immunoglobulins listed in the Biological Macromolecule Crystallization Database (Gilliland, G. L., Tung, M., Blakeslee, D. M. and Ladner, J. 1994. The Biological Macromolecule Crystallization Database, Version 3.0: New Features, Data, and the NASA Archive for Protein Crystal Growth Data. Acta Crystallogr. D50 408-413.) which have already been crystallised are principally Fab and Fc fragments.
WO02072636 describes antibody crystals, which, however, are prepared in a complex process with inoculation and using detergents, which should be avoided as far as possible in pharmaceutical formulations, and adjuvants, some of which are toxicologically unacceptable. In addition, the partide size cannot be controlled in the process described. In a control experiment (see Example 8), it was possible to show that the needle-shaped crystals described are obtained both from the protein solution and from the negative control (without protein) using the process described in WO02072636. It is clear from this that these are presumably at best protein inclusions in crystals of the precipitation reagent.
For the above-mentioned reasons, it is clear that crystallisation of anti-bodies is extremely difficult for the person skilled in the art and crystallisation processes disclosed in the literature cannot be applied to all known antibodies owing to the considerable heterogeneity of the different known antibodies with respect to primary, secondary and tertiary structure, glycosylation and structural flexibility. It was likewise unattractive to the person skilled in the art, for the above-mentioned reasons, to prepare precipitates of therapeutic antibodies since, in particular, irreversible denaturing was to be expected.
The object of the present invention was therefore to find stable forms, for example precipitates or crystals, for therapeutic proteins, in particular anti-bodies, so that their efficacy is retained during preparation, storage, transport and application. Since, as mentioned above, already established protein formulations generally cannot be applied to novel protein active ingredients, it was a further object of the present invention to find novel stable formulations for monoclonal antibodies against the EGF receptor, for example Mab C225 (cetuximab) and Mab h425 (EMD 72000). Although formulations comprising Mab C225 (cetuximab) or Mab h425 (EMD 72000) are disclosed in WO03053465 and WO 03/007988, the formulations disclosed in WO03053465 have, however, a relatively low protein concentration and they are not long-term-stable at room temperature, and the formulations disclosed in WO03007988 likewise have a relatively low protein concentration and the preparation (lyophilisate) has to be reconstituted before use. Consequently, a further object of the present invention was to find a stable pharmaceutical preparation which has a high concentration of the above-mentioned antibodies.
The process of lyophilisation for the stabilisation of protein formulations is disclosed, for example, in WO9300807 and WO9822136, but significant disadvantages of lyophilised preparations consist in that the user has to reconstitute the lyophilisate before use, which represents a considerable source of error in the preparation before use. Since a further preparation process is added compared with liquid formulations, the process is unfavourable with respect to additional work for process development (ensuring the stability during lyophilisation), preparation (preparation costs and duration) and, for example, validation.
The object of the present invention was thus to find solid forms and formulations for above-mentioned antibodies which have increased stability to stress conditions, such as elevated temperature, atmospheric humidity and/or shear forces, and comprise no toxicologically unacceptable adjuvants.